structural characterization of the n-terminal part of...
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192 http://dx.doi.org/10.1107/S2059798315024328 Acta Cryst. (2016). D72, 192–202
Received 5 October 2015
Accepted 17 December 2015
Edited by R. McKenna, University of
Florida, USA
Keywords: MERS-CoV; nucleocapsid; structure;
SAXS; RNA-binding domain.
PDB reference: N-terminal domain of the
MERS-CoV nucleocapsid, 4ud1
Supporting information: this article has
supporting information at journals.iucr.org/d
Structural characterization of the N-terminal partof the MERS-CoV nucleocapsid by X-ray diffractionand small-angle X-ray scattering
Nicolas Papageorgiou,a,b* Julie Lichiere,a,b Amal Baklouti,a,b Francois Ferron,a,b
Marion Sevajol,a,b Bruno Canarda,b and Bruno Coutarda,b*
aCNRS, AFMB UMR 7257, 13288 Marseille, France, and bAix-Marseille Universite, AFMB UMR 7257, 13288 Marseille,
France. *Correspondence e-mail: [email protected], [email protected]
The N protein of coronaviruses is a multifunctional protein that is organized into
several domains. The N-terminal part is composed of an intrinsically disordered
region (IDR) followed by a structured domain called the N-terminal domain
(NTD). In this study, the structure determination of the N-terminal region of the
MERS-CoV N protein via X-ray diffraction measurements is reported at a
resolution of 2.4 A. Since the first 30 amino acids were not resolved by X-ray
diffraction, the structural study was completed by a SAXS experiment to
propose a structural model including the IDR. This model presents the
N-terminal region of the MERS-CoV as a monomer that displays structural
features in common with other coronavirus NTDs.
1. Introduction
Middle East respiratory syndrome coronavirus (MERS-CoV)
is the aetiological agent responsible for an outbreak of a
severe respiratory syndrome disease in the Middle East with
sporadic spread to Europe, Africa, Asia and America. Since
June 2012, the date of the first clinical case observed in Saudi
Arabia, more than 1600 other cases have been confirmed, with
a fatality rate of 36%, as reported by the World Health
Organization in December 2015. In humans, MERS-CoV can
result in asymptomatic viraemia to severe respiratory distress
associated with organ failure. The animal source of the virus is
still under investigation, but to date scenarios involving bats
and/or camels have been proposed (Azhar et al., 2014; Corman
et al., 2014; Muller et al., 2015; Raj et al., 2014).
MERS-CoV is a positive-strand RNA virus belonging to the
Betacoronavirus genus within the Coronaviridae family of
the Nidovirales order. The 50 two-thirds of the MERS-CoV
genome contains open reading frames 1a and 1b, which are
translated into the viral polyproteins pp1a and pp1ab,
respectively. The processing of these two proteins leads to 16
nonstructural proteins which are involved in the replication
complex. The 30-proximal third encodes the viral structural
proteins and several accessory proteins, which are translated
from a set of subgenomic mRNAs. Among the structural
proteins, the nucleocapsid (N) protein is a key protein
displaying several functions. Indeed, it is not only involved in
genome packaging through the formation of a ribonucleo-
protein complex (RNP; reviewed in Chang et al., 2014), but it
also regulates viral RNA synthesis during replication and
transcription (Chang et al., 2014; Nelson et al., 2000; Stohlman
et al., 1988; Wu et al., 2014). Furthermore, the N protein
ISSN 2059-7983
# 2016 International Union of Crystallography
participates in deregulation of the cell cycle, host translational
shutoff and innate immunity disruption (reviewed in McBride
et al., 2014).
The coronavirus N protein is a >40 kDa protein organized
into two main folded domains called the N-terminal domain
(NTD) and the C-terminal domain (CTD) separated by a
flexible linker (Fig. 1a). The NTD is subdivided into an RNA-
binding module preceded by an intrinsically disordered region
(IDR). This IDR contributes to the RNA-binding properties
of the NTD (Chang et al., 2009). The linker region contains a
Ser/Arg stretch that could correspond to phosphorylation sites
involved in the regulation of the functions of the N protein
(Chang et al., 2009; He et al., 2004; Peng et al., 2008; Surjit et al.,
2005; Wu et al., 2009). The CTD is involved in N-protein
dimerization and RNA binding (Chen et al., 2007; Takeda et
al., 2008; Yu et al., 2006). It is followed by another IDR. Based
on electron microscopy or small-angle X-ray scattering data,
the proposed structural models of the N-protein organization
suggest that the NTD and CTD may be independent domains
with no interaction between each other (Chang et al., 2006).
Since the full-length N protein contains a large fraction
(>35%) of disordered regions that are recalcitrant to crystal-
lization, it is therefore relevant to study the structural and
functional features of each domain independently.
The three-dimensional structures of the NTDs from several
coronaviruses have previously been determined: those from
Severe acute respiratory syndrome coronavirus (SARS-CoV;
Huang et al., 2004; Saikatendu et al., 2007), infectious bron-
chitis virus Beaudette (IBV; Fan et al., 2005; Jayaram et al.,
2006), murine hepatitis virus (MHV; Grossoehme et al., 2009)
and human coronavirus OC43 (HCoV-OC43; Chen et al.,
2013). An alignment between the NTD sequences of MERS-
CoV, IBVand SARS-CoV shows 37% sequence identity, while
all reported coronavirus NTD structures are organized as a
five-stranded antiparallel �-sheet
core domain with disordered
loops of varying sizes connecting
the strands. A positively charged
�-hairpin protrudes out of the
core domain. Structural analysis
enabled this region to be
proposed as a possible RNA-
binding domain. Indeed, muta-
tional analysis and NMR studies
led to the conclusion that this
region is a major actor in RNA
recruitment (Chen et al., 2013;
Clarkson et al., 2009; Grossoehme
et al., 2009; Saikatendu et al.,
2007; Tan et al., 2006). Recently,
an inhibitor of the RNA–N
interaction has been demon-
strated to have an antiviral effect
on HCoV-OC43 (Lin et al., 2014).
The coronavirus N protein is thus
an attractive target for antiviral
research. Because of the world-
wide spread of MERS-CoV, with
potential risks for healthcare, it is
important to accumulate struc-
tural and functional information
on MERS-CoV proteins in order
to provide tools for antiviral
development.
In this study, we present the
three-dimensional structure of
the MERS-CoV NTD with its
upstream IDR region (hereafter
referred to as NTD+) at a reso-
lution of 2.4 A obtained by X-ray
diffraction measurements as well
as a small-angle X-ray scattering
(SAXS) study of the protein in
solution in order to assess the
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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 193
Figure 1Sequence analysis of MERS-CoV N protein. (a) Modular organization of the coronavirus N protein. Thedomain boundaries correspond to the MERS-CoV N protein numbering. IDR, intrinsically disorderedregion; NTD, N-terminal domain; CTD, C-terminal domain. (b) Sequence alignment of the N-terminalregion of several N proteins for which the structure of the NTD has been documented. The sequences arenamed as follows: virus acronym_PDB code. The depicted secondary structure was retrieved from theMERS-CoV NTD+ structure described in this study. The alignment was obtained using T-Coffee(Notredame et al., 2000) and ESPript 3.0 (Robert & Gouet, 2014).
oligomerization state of MERS-CoV NTD+, and propose a
structural model of NTD+ including its disordered region.
2. Materials and methods
2.1. Protein production, purification and characterization
The codon-optimized DNA encoding the N-terminal region
(amino acids 1–164; NTD+) of the MERS-CoV nucleocapsid
(strain Betacoronavirus England 1, accession No. KC164505)
was synthesized by GenScript. The DNA sequence was
amplified by a two-step PCR before cloning into the expres-
sion vector pMCOX20A to enable the fusion of a cleavable
thioredoxin/6�His tag at the N-terminus of NTD+ (Lantez et
al., 2011). The protein was then produced in Escherichia coli
T7 Express (DE3) cells (New England Biolabs). Expression
was induced overnight at 290 K in TB medium with 0.5 mM
IPTG when the OD600 nm of the culture had reached 0.6.
Purification of the protein and tag removal was performed in
non-denaturing conditions as described previously (Lantez et
al., 2011). The final size-exclusion chromatography (SEC) step
was performed in 10 mM HEPES, 300 mM NaCl pH 7.5. The
protein folding and stability were assessed by thermal shift
assays (Geerlof et al., 2006) following a previously described
protocol (Malet et al., 2009). The nonspecific RNA-binding
properties of the MERS-CoV NTD+ were assessed by fluor-
escence polarization-based assays using 50-CCAGGCGACA-
UCAGCG-30 RNA labelled at the 30 end with a Cy3 probe.
The binding assay was performed in 50 mM Tris buffer pH 7.5,
1 mM DTT, 2 mM MgCl2. The Cy3-labelled RNA was used
at 50 nM. Fluorescence polarization measurements were
performed in a microplate reader (PHERAstar FS, BMG
Labtech) with an optical module equipped with polarizers and
using excitation and emission wavelengths of 546 and 563 nm,
respectively.
In order to confirm the oligomerization state, analytical
SEC with online multi-angle laser light scattering, absorbance
and refractive index (MALLS/UV/RI) measurements were
carried out on an Alliance 2695 HPLC system (Waters) using a
Silica Gel KW802.5 column (Shodex) equilibrated in 10 mM
HEPES, 150 mM NaCl pH 7.5. Detection was performed using
a triple-angle light-scattering detector (miniDAWN TREOS,
Wyatt Technology), a quasi-elastic light-scattering instrument
(DynaPro, Wyatt Technology) and a differential refractometer
(Optilab rEX, Wyatt Technology). Molecular-weight and
hydrodynamic radius determination was performed by the
ASTRA V software (Wyatt Technology) using a dn/dc value of
0.185 ml g�1. Proteins were loaded at a final concentration of
5 mg ml�1.
2.2. Protein crystallization, data collection and processing
Crystallization trials were performed by the hanging-
drop vapour-diffusion method at 293 K using a nanodrop-
dispensing robot (Mosquito, TTP Labtech). Optimal crystal-
lization conditions were obtained by mixing 100 nl protein
solution at 25 mg ml�1 with 100 nl reservoir solution (0.1 M
sodium acetate pH 5, 2 M ammonium sulfate). The crystals of
MERS-CoV NTD+ were flash-cooled in liquid nitrogen at
110 K. Glycerol was added to the mother liquor as a cryo-
protectant to a final concentration of 20%.
Diffraction data for the native enzyme were collected on
the ID30A-1 beamline at the European Synchrotron Radia-
tion Facility (ESRF), Grenoble, France using a Pilatus3 2M
detector at a wavelength of 0.965 A and a temperature of
100 K.
The data were integrated by XDS (Kabsch, 2010), indexed
in space group C121 (unit-cell parameters a = 208.648,
b = 67.021, c = 60.94, � = � = � = 90), scaled and truncated to a
resolution of 2.4 A assuming a minimum accepted signal-to-
noise ratio equal to 2. The data were subsequently used for
molecular replacement with Phaser for phasing and were
refined and validated by PHENIX (Adams et al., 2010). The
molecular-graphics software Coot (Emsley & Cowtan, 2004)
was used for model building, while Chimera (Pettersen et al.,
2004) was used to visualize and draw the models.
2.3. Small-angle X-ray scattering (SAXS) measurements anddata processing
All SAXS measurements were carried out on beamline
BM29 at the ESRF at a working energy of 12.5 keV, leading to
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194 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202
Figure 2Biophysical characterization of MERS-CoV NTD+. (a) Binding of RNAto MERS-CoV NTD+ assessed by fluorescence polarization. 30-Cy5-labelled RNA was incubated with various concentrations of NTD+. (b)MALLS/UV/refractometry/SEC analysis of NTD+. The right y axisrepresents the OD280 nm and the left y axis represents the molar mass ing mol�1 (Da). The absorption peak is shown as a noncontinuous line, withthe continuous trace indicating the molar mass. The value of themeasured mass at the volume corresponding to the top of the peak isreported.
a scattering vector s ranging from 0.025 to 5 nm�1. Data were
recorded using a Pilatus 1M detector at a sample-to-detector
distance of 2.43 m. The scattering vector s is defined as
s ¼4�
�sin �; ð1Þ
where � is the wavelength of the incident radiation in nano-
metres and � is half of the angle between the incident and the
scattered radiation.
A range of seven protein concentrations was measured
(0.21, 0.41, 0.77, 1.55, 3.06, 5.85 and 11.66 mg ml�1) in 10 mM
HEPES pH 7.5, 300 mM NaCl. All samples were centrifuged
for 15 min at 15 000g before the experiment in order to
minimize contributions from aggregated particles.
The measurements proceeded with 45 ml protein sample
injected into a 1.8 mm capillary with a flow to minimize
radiation damage; data were collected at 277 K.
Ten exposures of 1 s each were made for each protein
concentration and were combined to give the average scat-
tering curve for each measurement. A buffer reference was
performed before and after measurement for each protein
sample in the same conditions. The forward scattering inten-
sity was calibrated using BSA as a reference at 5 mg ml�1.
Data were processed with the ATSAS package (Petoukhov
& Svergun, 2007) according to the standard procedure. Ten
frames of 1 s per protein were averaged using PRIMUS
(Konarev et al., 2003). Frames affected by radiation damage
were excluded and the buffer background was subtracted from
the sample.
3. Results
3.1. Protein production and purification
NTD+ of the MERS-CoV N protein was partly designed
based on available coronavirus N-protein crystal structures
(Chen et al., 2007; Fan et al., 2005; Grossoehme et al., 2009;
Saikatendu et al., 2007). Sequence alignments coupled with
disorder predictions revealed that the N-terminal intrinsically
disordered region (IDR) starts at position 1 and ends at
position 36, whereas the structured RNA-binding domain
(RBD) encompasses amino acids 37–164 (Fig. 1a). To date, the
crystal structures of NTDs from different coronaviruses have
been obtained after the removal of the IDR. In this study, the
domain encompassing amino acid 1 to amino acid 164 was
used to produce NTD+ of the MERS-CoV N, thus including
the N-terminal IDR. The recombinant wild-type (wt) protein
was produced in E. coli and was purified for crystallization,
SAXS experiments and oligomerization studies. The quality of
the recombinant protein was assessed by a thermal shift assay
(TSA) and an RNA-binding assay using a nonspecific RNA
sequence. TSA experiments revealed a transition curve from a
folded to an unfolded state for MERS-CoV NTD+, suggesting
that the protein is structured. Moreover, the observed melting
temperature (Tm) is 313 K. For comparison, the Tm of the
MERS-CoV NTD+ is 8 K lower than that obtained for the
SARS-CoV NTD (Fang et al., 2009). An RNA-binding assay
using fluorescence polarization was performed as an addi-
tional quality assessment. The protein displays an affinity
constant in the hundred-nanomolar range for a nonspecific
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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 195
Table 1X-ray data-collection and refinement statistics.
Data collectionWavelength (A) 0.965Resolution range (A) 48.26–2.48 (2.486–2.480)Space group C121Unit-cell parameters (A, �) a = 208.648, b = 67.021, c = 60.94,
� = 90, � = 89.98, � = 90Total reflections 101472 (9834)Unique reflections 32509 (3088)Multiplicity 3.2 (3.2)Completeness (%) 98.04 (97.87)Mean I/�(I) 5.23 (1.90)Wilson B factor (A2) 55.81Rmerge 0.143 (0.6257)Rmeas 0.1708CC1/2 0.967 (0.831)CC* 0.991 (0.953)
RefinementRwork 0.2281 (0.4445)Rfree 0.2738 (0.4771)No. of non-H atoms
Total 4922Macromolecules 4839Ligands 32Water 51
No. of protein residues 621R.m.s.d., bonds (A) 0.005R.m.s.d., angles (�) 0.87Ramachandran favoured (%) 98.2Ramachandran outliers (%) 0Rotamer outliers (%) 0C� outliers (%) 0Clashscore 5.08Average B factor (protein) (A2) 64.70Overall MolProbity score 1.27
Figure 3Ribbon representation of the coronavirus NTDs. Superposition of theMERS-CoV NTD+ structure (blue) with those of the IBV (green) and theSARS-CoV (red) NTDs, showing the relatively good superposition of themain core of the protein and the high flexibility of the interconnectingrandom-coil loops.
RNA, with a Kd of 153 nM. The results are shown in Fig. 2(a).
Together, these results show that MERS-CoV NTD+ is well
folded and suitable for crystallization.
3.2. Structure determination by X-ray diffraction
Molecular replacement (MR) was carried out using the IBV
NTD (PDB entry 2btl; Fan et al., 2005). A unique solution was
found in space group C121 with five molecules per asymmetric
unit. Subsequent refinement converged with no particular
problems to an Rwork of 0.23 and an Rfree of 0.27. Details of
data collection and refinement are shown in Table 1. The
structure was deposited as PDB entry 4ud1.
The NTD+ domain is globular overall (fully �), presenting a
kidney-shaped surface organized as a main core featuring
three antiparallel �-strands forming a central �-sheet from
which a flexible �-hairpin structure
extrudes. These features are inter-
connected with flexible coil loops. The
flexibility of these coils is reflected by
elevated B factors (overall versus local)
and is also illustrated in Fig. 3, where the
MERS-CoV NTD+ structure (PDB
entry 4ud1, blue) is superimposed with
the IBV (PDB entry 2btl; Fan et al.,
2005) and SARS-CoV (PDB entry 1ssk;
Huang et al., 2004) NTD structures
(green and red, respectively). The first
30 residues of the N-terminal region of
MERS-CoV NTD+, corresponding to a
disordered region, could not be
resolved. This region was previously
scarcely resolved in all structures
obtained using X-ray diffraction tech-
niques, while it was resolved as far as
amino acid 24 in the 1ssk structure
measured in solution by NMR.
Fig. 4 shows the electrostatic surfaces
of several N-terminal RNA-binding
domains of NTD+ originating from
closely related viruses. Surfaces were
calculated by APBS and truncated from
�5 mV (red) to +5 mV (blue). Figs. 4(a)
and 4(b) show front and back views of
the protein, respectively. The common
feature of all structures is the positively
charged hairpin (oriented towards the
top side in the figure) and a hydro-
phobic core domain (Fig. 5b). By
contrast, the bottom side presents some
differences in the charge distribution,
especially for the proteins originating
from murine hepatitis virus and human
coronavirus OC43, which show a more
important negatively charged region
compared with the other structures (Ma
et al., 2010).
Analyses of the molecular arrange-
ment within the crystal show stacking of
Pro33 in front of the Trp43 residue
belonging to the nearby molecule. The
same stacking is also found within the
crystal packing of the IBV NTD struc-
ture as presented in Fig. 5. In these
figures hydrophobic residues (Val, Ile,
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196 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202
Figure 4Front (a) and back (b) views of electrostatic surfaces from several NTDs originating from closelyrelated viruses. The structure of the SARS-CoV NTD was measured in solution by NMR, while theother structures were all obtained by single-crystal X-ray diffraction. Surfaces were calculated byAPBS and truncated from �5 mV (red) to +5 mV (blue).
Leu, Phe, Trp, Cys, Ala, Tyr, His, Thr, Ser, Gly and Lys)
following the classification of Livingstone & Barton (1993) are
labelled in green and highlight the conserved hydrophobic
propensity of the core domain.
3.3. Oligomerization assays
Preparative SEC experiments that were carried out for
protein crystallization and RNA binding revealed that wt
NTD+ eluted predominantly as monomers. However, this
result is not sufficient to claim that the protein cannot behave
as a transient multimer. SEC-MALLS experiments indicated
that the observed molecular weight was 18 kDa, which is close
to the theoretical molecular weight (17.8 kDa), confirming the
SEC results (Fig. 2b). The hydrodynamic radius (Rh) was
equal to 1.85 � 0.45 nm. Although the MALLS data exclude
the possibility that multimers are formed after the protein
elutes from SEC, cross-linking experiments were also
performed with NTD+. The results revealed that even in the
presence of glutaraldehyde NTD+ has a very poor ability to
produce cross-linked multimers (data not shown). Altogether,
the oligomerization studies suggest that NTD+ of the MERS-
CoV nucleocapsid behaves as a monomer in solution and that,
as suggested by the small interface between NTD+ molecules
in the crystal, the interaction observed in the crystal does not
reflect an interaction in solution. In order to characterize the
behaviour of MERS-CoV NTD+ in solution as well as to
obtain structural information about the N-terminal part of
NTD+, SAXS experiments were also carried out.
3.4. Small-angle X-ray scattering (SAXS) measurements
3.4.1. Guinier analysis. Fig. 6 shows the Guinier analysis in
the range sRg < 1.3, where Rg is the radius of gyration
expressed in nanometres (Rg = 2 nm). The data corresponding
to concentrations (conc) from 1.55 to 11.66 mg ml�1 present
increasing nonlinearity with concentration. Data are repre-
sented as open circles, while red lines correspond to a linear
regression. The values of concentration in mg ml�1, Rg in
nanometres as well as the normalized forward scattering
intensity I(0)/conc are shown. Both Rg
and I(0)/conc increase with increasing
concentration, as is characteristic of an
interparticle interference term origi-
nating from attractive intermolecular
forces. However, for the lower concen-
tration data (0.2, 0.41 and 0.77 mg ml�1)
the above values stabilize at approxi-
mately Rg = 2 nm and I(0)/conc = 26.5.
Taking into account the calibration
SAXS profile of BSA, we estimate a
molecular weight (MW) of 25 kDa,
which is approximately 30% higher than
the expected protein MW of 17.8 kDa.
3.4.2. GNOM analysis. All concen-
trations were processed separately by
GNOM (Svergun, 1992) using the
PRIMUS interface. The results are
shown in Table 2. All parameters
increase together with the concentra-
tion, as observed by Guinier analysis in
the low-s region. Taking into account a
mean value of the Porod volume (V)
calculated for the three lower concen-
trations, hVi = 27.7 nm3, we estimate an
MW in the range 13.8 < MW < 18.4 kDa
(V/2 < MW < V/1.5), which is in accor-
dance with the MW of the protein
monomer (17.8 kDa).
Considering the three lower concen-
trations, the GNOM analysis gives a
radius of gyration Rg of 2 nm, which is
equal to that found by the Guinier
analysis. The ratio of Rg and Rh (Rg/Rh)
provides shape information about
proteins. Comparison with the hydro-
dynamic radius Rh of 1.85 nm measured
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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 197
Figure 5Representations of the interaction between NTD molecules in the crystal. (a) Ribbonrepresentation of the structures at the interface between NTD+ of MERS-CoV (left panel) andthe NTD of IBV (right panel) in which Pro33 of the nearby molecule (in purple) is stacked withTrp43 (highlighted in red). (b) Surface representation of the same structures as in (a), wherehydrophobic residues are labelled in green and the others in grey.
by MALLS gives an Rg/Rh ratio of 1.08. The usual Rg/Rh value
for a globular protein is�0.775, which means that Rg is smaller
than Rh. However, when molecules deviate from globular to
nonspherical or elongated structures then Rg/Rh tends to
values above 0.775 as Rg becomes larger than Rh. In the
present case the Rg/Rh value is probably owing to a deviation
from a globular structure owing to the additional N-terminal
region (amino acids 1–30) of the protein which protrudes out
of the main globular domain of the protein as discussed below.
3.4.3. EOM analysis (ensemble-optimization method). A
preliminary analysis with DAMMIF (Franke & Svergun, 2009)
of all four lower concentrations separately showed almost
identical particles that nicely fit the experimental data (data
not shown). The calculated particles feature a globular region
with a protuberance (Fig. 10b). The structure 4ud1 described
above nicely fits the globular part. In order to model the
supplementary protuberant part of the DAMMIF particle we
considered an additional 30-amino-acid N-terminal region and
used the EOM 2.0 program (Bernado et al., 2007; Tria et al.,
2015). Model PDB structures with an N-terminal region
containing the missing sequence were created. Two different
kinds of EOM strategy were used: (i) the N-terminal region
(amino acids 1–30) was modelled as a fully random region and
(ii) the N-terminal region was divided into three equally long
fully random parts spanning amino acids 1–30. The globular
part of the protein taken from the 4ud1 structure was kept
fixed in all cases. Both strategies gave the same results with
respect to the final model structures as well as the corre-
sponding Rg, size distributions, Rflex and R� statistics given by
EOM.
EOM created a pool of 10 000 possible particles with Rg and
size distributed randomly over the Gaussian-like distribution
profiles shown in red bold lines in Fig. 7(a). For each specific
concentration data set EOM chooses particle ensembles
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198 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202
Figure 7EOM analysis. (a) Rg and size distributions for each concentration (blacksticks) calculated by EOM. Pool distributions are shown as redcontinuous lines. (b) Superposition of several representative ensemblemodels calculated by EOM.
Figure 6Guinier analysis in the range sRg < 1.3 (Rg = 2 nm). Experimental data arerepresented by open circles, while red lines correspond to a linearregression. The values of concentration (conc), Rg in nanometres as wellas the normalized forward scattering intensity I(0)/conc are shown.Both Rg and I(0)/conc increase with increasing concentration, which ischaracteristic of an interparticle interference term originating fromattractive intermolecular forces.
Table 2Results of GNOM and AUTOPOROD analysis of all concentrationsseparately.
Concentration(mg ml�1) Rg (nm) I(0)/conc
Porod volume(nm3) Dmax (nm)
0.20 2.04 26.8 27.1 8.00.41 2.06 26.8 28.8 8.50.77 2.13 29.5 27.0 8.51.55 2.21 30.7 30.2 8.53.06 2.40 32.3 29.7 10.95.85 2.65 38.0 32.7 11.611.66 3.01 32.2 36.1 14.0
containing all possible conformations that fit the data. The Rg
and size distributions of these ensembles are shown as black
sticks. Fig. 7(b) depicts the typical ensemble models calcu-
lated. Models with high Rg (>2.2 nm) and large size (>8 nm)
correspond to molecules with a spatially extended N-terminal
region, while lower values of Rg and size correspond to
molecules with an N-terminal region folded closely to the
main core.
In order to enable a quantitative characterization of the size
or Rg distributions, EOM uses two metrics: Rflex and R� (Tria et
al., 2015). The Rflex metric is given as a percentage in the range
0–100%, with Rflex = 100% indicating maximum flexibility. The
R� metric indicates the variance of the ensemble distribution
with respect to the original pool and is defined as the ratio
�e/�p, where �e and �p are the standard deviations for the
distributions of the selected ensemble and the pool, respec-
tively. Values close to 1 describe a fully flexible system.
In Table 3 we report the Rflex and R� metrics of the
ensembles, as well as Rflex-pool showing the Rflex metric of
the pool, as a function of concentration between 0.2 and
3.06 mg ml�1. For each concentration the Rflex of the ensemble
ranges between 75 and 85% with an Rflex-pool of 90% and a R�in the range between 0.99 and 1.3. These values suggest the
presence of important flexibility as discussed by Tria et al.
(2015). However, visual inspection of the Rg and size distri-
butions in Fig. 7 shows the presence of a main ensemble
population centred around Rg = 2.2 � 0.2 nm as well as the
presence of a secondary ensemble population centred around
Rg = 1.7 � 0.2 nm which is clearly evident for the three lower
concentrations. The presence of these distinct ensembles
might indicate two preferential conformations of the
N-terminal region, which could be open or closed.
3.4.4. Analysis of a merged profile. Taking into account all
the above analysis, the data at the five lower concentrations
(0.2, 0.41, 0.77, 1.55 and 3.06 mg ml�1) were merged in order
to retrieve a single data file with good signal to noise. The
low-s region of the merged file is an average of the 0.2 and
0.4 mg ml�1 data, while the high-s region is an average of the
0.77, 1.55 and 3.06 mg ml�1 data.
A full analysis of the merged data with GNOM, DAMMIF
and EOM was undertaken. Details of this analysis are shown
in Table 4. A three-dimensional ab initio model was
constructed from the merged SAXS data using DAMMIF (s
range from 0.1 to 5 nm�1).
No predefined shape or symmetry was used. 20 different
models were calculated and subsequently averaged and
filtered by DAMAVER (Volkov & Svergun, 2003). Fig. 8
presents the resulting numerical fits of GNOM, DAMMIF and
EOM analysis. The GNOM fit curve is completely super-
imposed on the DAMMIF fit curve shown by the red contin-
uous line, while the EOM fit curve is shown by the blue
research papers
Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 199
Figure 8GNOM, DAMMIF and EOM fits. The GNOM fit is completelysuperimposed on the DAMMIF fit shown by the red continuous line,while the EOM fit is shown by the blue continuous line together with themerged data (open circles). The error signals, red dots for GNOM andDAMMIF and blue dots for EOM, are shown at the bottom of the figureand they have been almost superimposed for spatial economy.
Table 4SAXS data-collection and scattering-derived parameters.
The scattering and molecular-mass parameters are related to the merged dataprofile discussed in the text.
Data-collection parametersInstrument BM29, ESRFDetector Pilatus 1MBeam geometry (mm) 700 � 700Wavelength (A) 0.992q range (nm�1) 0.025–5Exposure time (s) 1Concentration range (mg ml�1) 0.2–11.66Temperature (�C) 4
Structural parametersI(0)/conc from P(r) (arbitrary units) 26.8I(0)/conc from Guinier (arbitrary units) 26.5Rg from P(r) (nm) 2.04Rg from Guinier (nm) 1.95Dmax (nm) 8Porod volume (nm3) 27.5
Molecular-mass determinationPartial specific volume† (nm3) 21.4Molecular mass from I(0)‡ (kDa) 24.1Molecular mass from sequence (kDa) 17.731
Software employedPrimary data reduction FIT2DData processing ATSASAb initio analysis DAMMIFValidation and averaging DAMAVERComputation of intensities CRYSOLThree-dimensional graphics CHIMERA
† The partial specific volume was taken to be (1.21 � MW) A3 per molecule (Harpaz etal., 1994). ‡ The molecular weight was calculated using calibrated data for BSA(66 kDa) at a concentration of 5 mg ml�1.
Table 3Rflex, Rflex-pool and R� metrics as a function of the concentration calculatedby EOM for the five lower concentrations separately.
Concentration (mg ml�1) Rflex (%) Rflex-pool (%) R�
0.20 76.8 91.1 0.990.41 75.1 90.4 1.300.77 78.8 90.5 1.201.55 84.7 90.1 0.953.06 85.2 89.4 1.32
continuous line along with the merged data (open circles). The
error signals, red dots for GNOM and DAMMIF and blue dots
for EOM, are shown at the bottom of the figure and have been
almost superimposed for spatial economy.
Fig. 9(a) shows the P(r) distribution of the merged data with
Dmax = 8 nm. Fig. 9(b) shows a Kratky plot of the merged data,
featuring an increase in the signal in the high-s region
(>2.5 nm�1), probably owing to the flexible N-terminal region.
In Fig. 10(a) we show the three ensemble structures
resulting from the EOM analysis of the merged data together
with the corresponding Rg and size distributions considered by
EOM (Fig. 10b). The contribution of each ensemble to the
measured SAXS profile is shown as a percentage under each
ensemble structure.
The corresponding metrics Rflex = 84% (Rflex-pool = 91%)
and R� = 1.12 correspond to a predominantly flexible
N-terminal region, while the size and Rg distributions shown
in Fig. 10(b) provide evidence for two distinct populations
(around Rg = 2.2 nm and Rg = 1.6 nm) as calculated previously
for each concentration separately. As a conclusion from the
EOM analysis, we can stress the presence of a flexible
N-terminal region together with the presence of two confor-
mations for the molecule: open and closed. In all cases the first
20 amino acids form a loop without being randomly unfolded.
The three ensemble model structures calculated by EOM fit
within the filtered particle calculated by DAMAVER as shown
in Fig. 10(c). The protuberant part of the particle mainly
corresponds to the open conformation with the N-terminal
region pointing out from the globular part.
4. Discussion and conclusion
MERS NTD+ encompassing amino acids 1–164 was crystal-
lized even though sequence prediction revealed the presence
of a disordered N-terminal region from amino acid 1 to amino
acid 37. Attempts to crystallize the NTD domain (from
positions 37 to 164) did not lead to suitable crystals from
commercial screens. It should be noted that during the writing
of this study a preliminary crystallographic analysis of the
NTD region encompassing amino acids 39–165 has been
published (Wang et al., 2015).
Our structural study therefore focused on the NTD+
domain, which could provide additional structural information
by combining crystal structure and SAXS analysis. Interest-
ingly, the asymmetric unit of the crystal was made up of five
NTD+ molecules organized with an interaction between Pro33
and Trp43 of the neighbouring molecule (Fig. 5). This inter-
action highlights the role of the intrinsically disordered region
(IDR) in the crystal organization and illustrates the need to
design several constructs for a given domain of interest in
order to improve the success rate at the level of protein
expression or crystallization (Bignon et al., 2013; Graslund et
al., 2008).
Although divergent in size and sequence, the NTDs for
which structures have been determined to date share a
common fold with a central �-sheet and a �-hairpin extension.
As expected, the three-dimensional structure of MERS-CoV
NTD+ displays the same structural organization. Its �-exten-
sion is positively charged (Fig. 4) and in several coronaviruses
it has been proposed to contribute to RNA binding through
electrostatic interactions with the phosphodiester groups of
the RNA (Fan et al., 2005). In addition to these interactions,
the conserved hydrophobic core domain could interact with
bases of the RNA through several aromatic residues (Huang
et al., 2004; McBride et al., 2014; Spencer & Hiscox, 2006).
Interestingly, in the crystal structure of MERS-CoV NTD+,
Trp43 located in the core domain was found to stack with a
proline (Pro33) of the adjacent NTD+, as in the crystal packing
of IBV NTD (Huang et al., 2004).
We therefore wanted to assess the oligomerization state
of MERS-CoV NTD+. SEC-MALLS analysis together with
SAXS data demonstrated that, at least under our experi-
mental conditions at pH 7.5, MERS NTD+ behaves mainly as a
monomer in solution. As previously reported, the oligomer-
ization of the MHV N protein is partly mediated by an
interaction between the NTD modules (Hurst et al., 2009).
Moreover, the NTD of the IBV N protein also has a weak
research papers
200 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202
Figure 9Distance distribution and Kratky plot. (a) Distance distribution P(r) ofthe merged data with Dmax = 8 nm. (b) Kratky plot of the merged datashowing the characteristic increase of the signal in the high-s region(greater than 2.5 nm�1) owing to partial disorder of the molecule.
propensity for dimerization (Fan et al., 2005; Jayaram et al.,
2006). We can thus propose that, by itself, MERS-CoV NTD+
cannot promote MERS-CoV N-protein assembly, but could
contribute by a mechanism that still has to be unravelled to the
formation of the RNP through NTD–NTD interaction after
the CTD has already promoted the oligomerization.
The role of Trp43 in RNA binding has also been addressed.
MERS-CoV NTD+ binds to a nonspecific RNA in the
hundred-nanomolar range, and a W43A substitution did not
significantly alter the affinity constant (data not shown). RNA-
binding studies on MHV NTD revealed that aromatic residues
located in �3 and �5 are strong determinants for sequence-
specific RNA binding (Grossoehme et
al., 2009), whereas Trp43 precedes the
�1 strand. However, analysis of RNA
binding to the SARS-CoV NTD by
NMR showed that even for a poly-
adenine RNA the core region is still
involved in the binding (Huang et al.,
2004). It is therefore possible that
Trp43 could be involved in RNA
binding, but not as a main contributor,
and its single substitution does not allow
perturbation of the function. At this
stage, the role of Trp43, a conserved
residue in the betacoronaviruses, still
remains to be investigated.
SAXS experiments were undertaken
to characterize MERS-CoV NTD+ in
solution. The SAXS results showed
that the protein is monomeric and
composed of a globular part and a
flexible N-terminal region of 30 resi-
dues. This region is not completely
disordered but points out from the
globular part with two preferential
conformations: an open conformation
and a closed conformation. In all cases
the first 20 amino acids form a loop
without being randomly unfolded. Let
us recall that this N-terminus is involved
in RNA binding, working cooperatively
with the other RNA-binding sites of the
N protein (Chang et al., 2009). However,
it is possible that it plays other roles
since viral IDRs tend to be involved in
various interactions with host-cell
components other than RNA, such as
host membranes or proteins (Xue et al.,
2014).
Acknowledgements
This work was supported by the
SILVER Large Scale Collaborative
Project (grant agreement No. 260644) of
the European Union Seventh Frame-
work and the French Infrastructure for
Integrated Structural Biology (FRISBI)
ANR-10-INSB-05-01. The authors also
thank the ESRF for provision of
synchrotron beam time and the
MASSIF and BM29 staff for data
collection, as well as Stephanie Blangy
research papers
Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 201
Figure 10EOM analysis of a merged data set. (a) Three ensemble model structures resulting from EOManalysis of the merged data together with the corresponding percentage presence. (b) Rg and sizedistributions of the merged data. (c) Fit of the three structures within the filtered DAMAVERparticle.
and Silvia Spinelli for their contribution to the
experiments.
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202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202